C1-inhibitor influence on platelet activation by thrombin receptors agonists.

INTRODUCTION: Protease activated receptors 1 (PAR1) and 4 (PAR4) agonists are used to study platelet activation. Data on platelet activation are extrapolated across experimental settings. C1-inhibitor (C1INH) is a protease inhibitor present in plasma but not in isolated platelet suspensions. Here we show that C1INH affects platelet activation through PAR1 and PAR4 agonists. METHODS: Platelets were isolated from healthy donor whole blood and then labeled with anti-CD62P and PAC1 antibodies. The platelet suspensions were exposed to PAR1 agonists SFLLRN, TFLLR and TFLLRN; PAR4 agonists AYPGKF and GYPGQV; ADP and thrombin. Flow-cytometric measurements were performed in 5, 10 and 15 min after activation. RESULTS: 0.25 mg/ml C1INH addition made platelets to faster expose CD62P and glycoprotein IIb/IIIa complex after activation with PAR1 agonists. Conversely, C1INH addition led to inhibition of platelet activation with PAR4 agonists and thrombin. Activation with ADP was not affected by C1INH. CONCLUSIONS: Our results suggest that C1INH can modify platelet activation in the presence of synthetic PAR agonists used in platelet research. These observations may be relevant to the development of new methods to assess platelet function.

Protease Activated Receptors and Arthritis.

The catabolic and destructive activity of serine proteases in arthritic joints is well known; however, these enzymes can also signal pain and inflammation in joints. For example, thrombin, trypsin, tryptase, and neutrophil elastase cleave the extracellular N-terminus of a family of G protein-coupled receptors and the remaining tethered ligand sequence then binds to the same receptor to initiate a series of molecular signalling processes. These protease activated receptors (PARs) pervade multiple tissues and cells throughout joints where they have the potential to regulate joint homeostasis. Overall, joint PARs contribute to pain, inflammation, and structural integrity by altering vascular reactivity, nociceptor sensitivity, and tissue remodelling. This review highlights the therapeutic potential of targeting PARs to alleviate the pain and destructive nature of elevated proteases in various arthritic conditions.

[Downregulation of proteinase activated receptor 4 inhibits migration of SW620 human colorectal cancer cells].

OBJECTIVE: To establish the human colorectal cancer cell model SW620/PAR4D with inducible suppression of proteinase activated receptor 4 (PAR4) expression, and investigate the role PAR4 plays in the proliferation and migration of cancer cells. METHODS: A human colorectal cancer cell line with tetracycline-inducible expression regulatory system, namely SW620/Tet-on, was established; inducible expression lentiviral vector with artificial microRNA targeting PAR4, pLVX-Tight-Puro-PAR4-miR, was constructed and transfected into SW620/Tet-on to make an inducible PAR4-suppressed cell model SW620/PAR4D. Western blotting was used to confirm the suppression of PAR4 expression after the doxycycline (DOX) treatment. CCK-8 assay was used to evaluate the impact of suppressed PAR4 expression on cell proliferation, and wound-healing assay was used to analyze the migration of the cells. RESULTS: The SW620/PAR4D cell model was established successfully. Suppression of PAR4 expression by DOX treatment had no significant impact on the growth/proliferation of SW620/PAR4D cells, but markedly inhibited the cell migration. CONCLUSION: Suppression of PAR4 expression has no significant effect on the proliferation of SW620 cells, but can inhibit the migration of the cells.

Thrombin-induced reactive oxygen species generation in platelets: A novel role for protease-activated receptor 4 and GPIbα.

BACKGROUND: Platelets are essential for maintaining haemostasis and play a key role in the pathogenesis of cardiovascular disease. Upon ligation of platelet receptors through subendothelial matrix proteins, intracellular reactive oxygen species (ROS) are generated, further amplifying the platelet activation response. Thrombin, a potent platelet activator, can signal through GPIbα and protease-activated receptor (PAR) 1 and PAR4 on human platelets, and recently has been implicated in the generation of ROS. While ROS are known to have key roles in intra-platelet signalling and subsequent platelet activation, the precise receptors and signalling pathways involved in thrombin-induced ROS generation have yet to be fully elucidated. OBJECTIVE: To investigate the relative contribution of platelet GPIbα and PARs to thrombin-induced reactive oxygen species (ROS) generation. METHODS AND RESULTS: Highly specific antagonists targeting PAR1 and PAR4, and the GPIbα-cleaving enzyme, Naja kaouthia (Nk) protease, were used in quantitative flow cytometry assays of thrombin-induced ROS production. Antagonists of PAR4 but not PAR1, inhibited thrombin-derived ROS generation. Removal of the GPIbα ligand binding region attenuated PAR4-induced and completely inhibited thrombin-induced ROS formation. Similarly, PAR4 deficiency in mice abolished thrombin-induced ROS generation. Additionally, GPIbα and PAR4-dependent ROS formation were shown to be mediated through focal adhesion kinase (FAK) and NADPH oxidase 1 (NOX1) proteins. CONCLUSIONS: Both GPIbα and PAR4 are required for thrombin-induced ROS formation, suggesting a novel functional cooperation between GPIbα and PAR4. Our study identifies a novel role for PAR4 in mediating thrombin-induced ROS production that was not shared by PAR1. This suggests an independent signalling pathway in platelet activation that may be targeted therapeutically.

Polidocanol inhibits cowhage - but not histamine-induced itch in humans.

Polidocanol is a local anaesthetic and antipruritic compound that is used in the treatment of itching skin conditions such as eczema. Its mechanisms of action are largely ill defined. This study has compared the antipruritic efficacy of topical polidocanol in histamine-induced itch and a histamine-independent, cowhage-induced model of pruritus. Polidocanol (3%) or vehicle was applied topically under occlusion for 1 h to the forearms of 45 healthy volunteers before itch was provoked by rubbing in 40-45 spicules of cowhage or skin prick testing with 10 mg/ml histamine. Itch was recorded at 1-min intervals for 30 min on a 100-mm visual analogue scale. Polidocanol significantly reduced the area under the curve for cowhage-induced itch by 58% (P < 0.05), but had no significant effect on histamine-induced itch. This result underlines the importance of histamine-independent itch models in the development of topical antipruritic agents.

Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation, platelet aggregation, and P-selectin expression.

In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.

Targeting platelet migration in the postischemic liver by blocking protease-activated receptor 4.

BACKGROUND: Platelets play a critical role during hepatic ischemia/reperfusion (I/R). Antiplatelet strategies during liver transplantation are, however, limited because of bleeding complications. Thrombin is activated during reperfusion and regulates platelet and endothelial cell function via protease-activated receptor 4 (PAR-4). Interventions at the level of PAR-4, the main platelet receptor for thrombin, are assumed to attenuate the proinflammatory effects of thrombin without affecting blood coagulation. The aim of our study was to analyze the impact of PAR-4 blockade on platelet recruitment and microvascular injury during hepatic I/R. METHODS: C57BL/6 mice undergoing hepatic I/R (90 min/60 min and 240 min) were treated either with a selective PAR-4 antagonist TcY-NH2 or vehicle. Sham-operated animals served as controls. Recruitment of freshly isolated and fluorescence-labeled platelets and CD4 T cells was analyzed using intravital video fluorescence microscopy. Parameters of tissue injury, regeneration, and blood coagulation were assessed in tissue/blood samples. RESULTS: Results show that treatment with TcY-NH2 attenuated I/R-induced platelet and CD4 T-cell recruitment, improved sinusoidal perfusion failure, and reduced apoptotic and necrotic injury. The protective effect of PAR-4 blockade did not suppress hemostasis or liver regeneration. CONCLUSION: Our in vivo data suggest PAR-4 as a potential target for future therapeutic strategies against platelet-mediated liver injury on transplantation.

Increased responsiveness to thrombin through protease-activated receptors (PAR)-1 and -4 in active Crohn's disease.

BACKGROUND AND AIMS: Platelets are essential in hemostasis and inflammation, thereby linking coagulation with inflammation. Abundant thrombin generation in association with inflammation is considered a major reason for the increased risk for thromboembolic events. We therefore investigated platelet responsiveness to thrombin. METHODS: In this case-control study 85 patients with Crohn's disease (active CD 42, remission 43) and 30 sex- and age-matched controls were enrolled. Clinical disease activity (Harvey-Bradshaw-Index) was assessed and CD-related data were determined by chart review. Platelets' response to protease activated receptor-1 and -4 (PAR-1, -4) was assessed by whole blood platelet aggregometry (MEA), levels of platelets adhering to monocytes (PMA), and platelet surface P-selectin. RESULTS: Platelets' aggregation after activation with the specific PAR-1 agonist (SFLLRN) and PAR-4 agonist (AYPGKF) was higher in patients with active CD compared to patients in remission and controls (p=0.0068 and p=0.0023 for SFLLRN, p=0.0019 and 0.0003 for AYPGKF). Likewise, levels of PMA after activation with PAR-1 and PAR-4 receptor agonists were higher in patients with active CD compared to patients in remission and controls (p=0.0001 and p<0.0001 for SFLLRN, p=0.0329 and p=0.0125 for AYPGKF). However, P-selectin expression on human platelets showed heterogeneous results. Only PAR-1 activation of platelets resulted in significant differences between CD patients and controls (p=0.0001 and p=0.0022 for active and inactive CD versus controls, respectively). CONCLUSIONS: Our data suggest a new mechanism of platelet activation which has the potential to increase risk for thromboembolism in patients with active CD which might be due to platelets poised for thrombin-inducible activation.

Novel mechanisms for activated protein C cytoprotective activities involving noncanonical activation of protease-activated receptor 3.

The direct cytoprotective activities of activated protein C (APC) on cells convey therapeutic, relevant, beneficial effects in injury and disease models in vivo and require the endothelial protein C receptor (EPCR) and protease activated receptor 1 (PAR1). Thrombin also activates PAR1, but its effects on cells contrast APC's cytoprotective effects. To gain insights into mechanisms for these contrasting cellular effects, protease activated receptor 3 (PAR3) activation by APC and thrombin was studied. APC cleaved PAR3 on transfected and endothelial cells in the presence of EPCR. Remarkably, APC cleaved a synthetic PAR3 N-terminal peptide at Arg41, whereas thrombin cleaved at Lys38. On cells, APC failed to cleave R41Q-PAR3, whereas K38Q-PAR3 was still cleaved by APC but not by thrombin. PAR3 tethered-ligand peptides beginning at amino acid 42, but not those beginning at amino acid 39, conveyed endothelial barrier-protective effects. In vivo, the APC-derived PAR3 tethered-ligand peptide, but not the thrombin-derived PAR3 peptide, blunted vascular endothelial growth factor (VEGF)-induced vascular permeability. These data indicate that PAR3 cleavage by APC at Arg41 can initiate distinctive APC-like cytoprotective effects. These novel insights help explain the differentiation of APC's cytoprotective versus thrombin's proinflammatory effects on cells and suggest a unique contributory role for PAR3 in the complex mechanisms underlying APC cytoprotective effects.

Protease-activated receptor (PAR) 1 and PAR4 differentially regulate factor V expression from human platelets.

With the recent interest of protease-activated receptors (PAR) 1 and PAR4 as possible targets for the treatment of thrombotic disorders, we compared the efficacy of protease-activated receptor (PAR)1 and PAR4 in the generation of procoagulant phenotypes on platelet membranes. PAR4-activating peptide (AP)-stimulated platelets promoted thrombin generation in plasma up to 5 minutes earlier than PAR1-AP-stimulated platelets. PAR4-AP-mediated factor V (FV) association with the platelet surface was 1.6-fold greater than for PAR1-AP. Moreover, PAR4 stimulation resulted in a 3-fold greater release of microparticles, compared with PAR1 stimulation. More robust FV secretion and microparticle generation with PAR4-AP was attributable to stronger and more sustained phosphorylation of myosin light chain at serine 19 and threonine 18. Inhibition of Rho-kinase reduced PAR4-AP-mediated FV secretion and microparticle generation to PAR1-AP-mediated levels. Thrombin generation assays measuring prothrombinase complex activity demonstrated 1.5-fold higher peak thrombin levels on PAR4-AP-stimulated platelets, compared with PAR1-AP-stimulated platelets. Rho-kinase inhibition reduced PAR4-AP-mediated peak thrombin generation by 25% but had no significant effect on PAR1-AP-mediated thrombin generation. In conclusion, stimulation of PAR4 on platelets leads to faster and more robust thrombin generation, compared with PAR1 stimulation. The greater procoagulant potential is related to more efficient FV release from intracellular stores and microparticle production driven by stronger and more sustained myosin light chain phosphorylation. These data have implications about the role of PAR4 during hemostasis and are clinically relevant in light of recent efforts to develop PAR antagonists to treat thrombotic disorders.

Protease-activated receptor-1 inhibitors: a novel class of antiplatelet agents for the treatment of patients with acute coronary syndrome.

The unifying basis of acute coronary syndrome (ACS) is the complication of a vulnerable coronary plaque, an event primarily mediated by platelet activation. Three major pathways are predominantly involved in this process: thromboxane A(2) via the thromboxane A(2) receptor, adenosine diphosphate via the P2Y(12) receptor, and thrombin via the protease-activated receptor (PAR)-1, with the latter being the most potent platelet activator. Despite the effective inhibition of the first two pathways with aspirin and an expanding family of P2Y(12) inhibitors, respectively, the recurrence of ischemic events in patients with ACS remains high. There is also a growing concern regarding the safety profile in terms of bleeding with more powerful antiplatelet agents, which has tempered expectations of newly developed compounds. PAR-1 inhibitors are a novel class of antiplatelet agents that inhibit thrombin-mediated platelet activation. Preliminary data indicate that these compounds have the potential to improve ischemic prognosis without increasing the bleeding risk. In this chapter we will discuss the rationale for developing this novel class of antiplatelet agents and specifically, the two compounds in most advanced clinical development, vorapaxar and atopaxar.

Thrombin induces heme oxygenase-1 expression in human synovial fibroblasts through protease-activated receptor signaling pathways.

INTRODUCTION: Thrombin is a key factor in the stimulation of fibrin deposition, angiogenesis, and proinflammatory processes. Abnormalities in these processes are primary features of osteoarthritis (OA). Heme oxygenase (HO)-1 is a stress-inducible rate-limiting enzyme in heme degradation that confers cytoprotection against oxidative injury. Here, we investigated the intracellular signaling pathways involved in thrombin-induced HO-1 expression in human synovial fibroblasts (SFs). METHODS: Thrombin-mediated HO-1 expression was assessed with quantitative real-time (q)PCR. The mechanisms of action of thrombin in different signaling pathways were studied by using Western blotting. Knockdown of protease-activated receptor (PAR) proteins was achieved by transfection with siRNA. Chromatin immunoprecipitation assays were used to study in vivo binding of Nrf2 to the HO-1 promoter. Transient transfection was used to examine HO-1 activity. RESULTS: Osteoarthritis synovial fibroblasts (OASFs) showed significant expression of thrombin, and expression was higher than in normal SFs. OASFs stimulation with thrombin induced concentration- and time-dependent increases in HO-1 expression. Pharmacologic inhibitors or activators and genetic inhibition by siRNA of protease-activated receptors (PARs) revealed that the PAR1 and PAR3 receptors, but not the PAR4 receptor, are involved in thrombin-mediated upregulation of HO-1. Thrombin-mediated HO-1 expression was attenuated by thrombin inhibitor (PPACK), PKCδ inhibitor (rottlerin), or c-Src inhibitor (PP2). Stimulation of cells with thrombin increased PKCδ, c-Src, and Nrf2 activation. CONCLUSION: Our results suggest that the interaction between thrombin and PAR1/PAR3 increases HO-1 expression in human synovial fibroblasts through the PKCδ, c-Src, and Nrf2 signaling pathways.

Mouse model of touch-evoked itch (alloknesis).

Lightly touching normal skin near a site of itch can elicit itch sensation, a phenomenon known as alloknesis. To investigate the neural mechanisms of alloknesis, we have developed an animal model. Low-threshold mechanical stimulation of the skin normally does not elicit any response in naive C57/BL6 mice. Following acute intradermal (i.d.) injection of histamine in the rostral back, mechanical stimulation 7 mm from the injection site elicited discrete hindlimb scratch bouts directed toward the stimulus. This began at 10 minutes and peaked 20-40 minutes post histamine injection, declining over the next hour. Histamine itself elicited bouts of scratching not associated with the mechanical stimulus, which ceased after 30 minutes. Histamine- and touch-evoked scratching was inhibited by the µ-opiate antagonist naltrexone. Touch-evoked scratching was observed following i.d. 5-HT (5-hydroxytryptamine), a protease-activated receptor (PAR)-4 agonist, and an MrgprC11 agonist BAM8-22, but not chloroquine or a PAR-2 agonist. The histamine H1 receptor antagonist terfenadine prevented scratching and alloknesis evoked by histamine, but not 5-HT, a PAR-4 agonist or an MrgprC11 agonist. In mice with experimental dry skin, there was a time-dependent increase in spontaneous and touch-evoked scratching. This animal model appears to be useful to investigate neural mechanisms of itch and alloknesis.

Thrombin receptor levels in platelet concentrates during storage and their impact on platelet functionality.

BACKGROUND: Quality control of platelet (PLT) concentrates is challenging, due to PLT lesions, which are difficult to detect with routine methods. The search for reliable PLT lesion biomarkers is focused on the role of PLTs in primary hemostasis. PLT transfusions also have a significant impact on secondary hemostasis. In this phase, responsiveness of PLTs to small amounts of thrombin is crucial. PAR1 and PAR4 are protease-activated receptors and are responsible for thrombin reactivity of human PLTs. This study should elucidate if levels of those two receptors are changing in PLT concentrates during storage and if those changes have an impact on PLT aggregation and support of thrombin generation. STUDY DESIGN AND METHODS: PLT concentrates from buffy coat preparations were stored in SSP+ solution for 9 days at 22±2°C on a horizontal flatbed agitator, and samples were taken daily for analysis. PAR1 and PAR4 levels were evaluated using Western blot analysis. PLT aggregation was measured using Born aggregometry and specific PAR1 or PAR4 agonists. Thrombin generation was measured using calibrated automated thrombography. RESULTS: Levels of both receptors (PAR1 and PAR4) started to decrease after 5 days of storage. PAR1-mediated PLT aggregation remained constant, whereas PAR4-mediated PLT aggregation decreased with storage time. Rate of thrombin generation was accelerated after 5 days of storage. CONCLUSION: Decreasing levels of PARs in PLT concentrates after 5 days of storage influenced PAR4-mediated, but not PAR1-mediated, aggregation. Thrombin generation with senescent PLTs was increased, which may be attributed to other mechanisms promoting increased phosphatidylserine exposure.

Solulin reduces infarct volume and regulates gene-expression in transient middle cerebral artery occlusion in rats.

BACKGROUND: Thrombolysis after acute ischemic stroke has only proven to be beneficial in a subset of patients. The soluble recombinant analogue of human thrombomodulin, Solulin, was studied in an in vivo rat model of acute ischemic stroke. METHODS: Male SD rats were subjected to 2 hrs of transient middle cerebral artery occlusion (tMCAO). Rats treated with Solulin intravenously shortly before reperfusion were compared to rats receiving normal saline i.v. with respect to infarct volumes, neurological deficits and mortality. Gene expression of IL-6, IL-1ß, TNF-α, MMP-9, CD11B and GFAP were semiquantitatively analyzed by rtPCR of the penumbra. RESULTS: 24 hrs after reperfusion, rats were neurologically tested, euthanized and infarct volumes determined. Solulin significantly reduced mean total (p=0.001), cortical (p=0.002), and basal ganglia (p=0.036) infarct volumes. Hippocampal infarct volumes (p=0.191) were not significantly affected. Solulin significantly downregulated the expression of IL-1ß (79%; p<0.001), TNF-α (59%; p=0.001), IL-6 (47%; p=0.04), and CD11B (49%; p=0.001) in the infarcted cortex compared to controls. CONCLUSIONS: Solulin reduced mean total, cortical and basal ganglia infarct volumes and regulated a subset of cytokines and proteases after tMCAO suggesting the potency of this compound for therapeutic interventions.

Protein kinase Cδ mediates the activation of protein kinase D2 in platelets.

Protein kinase D (PKD) is a subfamily of serine/threonine specific family of kinases, comprised of PKD1, PKD2 and PKD3 (PKCµ, PKD2 and PKCv in humans). It is known that PKCs activate PKD, but the relative expression of isoforms of PKD or the specific PKC isoform/s responsible for its activation in platelets is not known. This study is aimed at investigating the pathway involved in activation of PKD in platelets. We show that PKD2 is the major isoform of PKD that is expressed in human as well as murine platelets but not PKD1 or PKD3. PKD2 activation induced by AYPGKF was abolished with a G(q) inhibitor YM-254890, but was not affected by Y-27632, a RhoA/p160ROCK inhibitor, indicating that PKD2 activation is G(q)-, but not G12/13-mediated Rho-kinase dependent. Calcium-mediated signals are also required for activation of PKD2 as dimethyl BAPTA inhibited its phosphorylation. GF109203X, a pan PKC inhibitor abolished PKD2 phosphorylation but Go6976, a classical PKC inhibitor had no effect suggesting that novel PKC isoforms are involved in PKD2 activation. Importantly, Rottlerin, a non-selective PKCδ inhibitor, inhibited AYPGKF-induced PKD2 activation in human platelets. Similarly, AYPGKF- and Convulxin-induced PKD2 phosphorylation was dramatically inhibited in PKCδ-deficient platelets, but not in PKCθ- or PKCɛ-deficient murine platelets compared to that of wild type platelets. Hence, we conclude that PKD2 is a common signaling target downstream of various agonist receptors in platelets and G(q)-mediated signals along with calcium and novel PKC isoforms, in particular, PKCδ activate PKD2 in platelets.

Regulation of human vascular protease-activated receptor-3 through mRNA stabilization and the transcription factor nuclear factor of activated T cells (NFAT).

Thrombin promotes vascular smooth muscle cell (SMC) proliferation and inflammation via protease-activated receptor (PAR)-1. A further thrombin receptor, PAR-3, acts as a PAR-1 cofactor in some cell-types. Unlike PAR-1, PAR-3 is dynamically regulated at the mRNA level in thrombin-stimulated SMC. This study investigated the mechanisms controlling PAR-3 expression. In human vascular SMC, PAR-3 siRNA attenuated thrombin-stimulated interleukin-6 expression and extracellular signal-regulated kinases 1/2 phosphorylation, indicating PAR-3 contributes to net thrombin responses in these cells. Thrombin slowed the decay of PAR-3 but not PAR-1 mRNA in the presence of actinomycin D and induced cytosolic shuttling and PAR-3 mRNA binding of the mRNA-stabilizing protein human antigen R (HuR). HuR siRNA prevented thrombin-induced PAR-3 expression. By contrast, forskolin inhibited HuR shuttling and destabilized PAR-3 mRNA, thus reducing PAR-3 mRNA and protein expression. Other cAMP-elevating agents, including the prostacyclin-mimetic iloprost, also down-regulated PAR-3, accompanied by decreased HuR/PAR-3 mRNA binding. Iloprost-induced suppression of PAR-3 was reversed with a myristoylated inhibitor of protein kinase A and mimicked by phorbol ester, an inducer of cyclooxygenase-2. In separate studies, iloprost attenuated PAR-3 promoter activity and prevented binding of nuclear factor of activated T cells (NFAT2) to the human PAR-3 promoter in a chromatin immunoprecipitation assay. Accordingly, PAR-3 expression was suppressed by the NFAT inhibitor cyclosporine A or NFAT2 siRNA. Thus human PAR-3, unlike PAR-1, is regulated post-transcriptionally via the mRNA-stabilizing factor HuR, whereas transcriptional control involves NFAT2. Through modulation of PAR-3 expression, prostacyclin and NFAT inhibitors may limit proliferative and inflammatory responses to thrombin after vessel injury.

Intracolonic infusion of fecal supernatants from ulcerative colitis patients triggers altered permeability and inflammation in mice: role of cathepsin G and protease-activated receptor-4.

BACKGROUND: Cathepsin G (Cat-G) is a neutrophil serine-protease found in the colonic lumen of ulcerative colitis (UC) patients. Cat-G is able to activate protease-activated receptor-4 (PAR(4) ) located at the apical side of enterocytes, leading to epithelial barrier disruption. However, the mechanisms through which Cat-G triggers inflammation are not fully elucidated. The aims of our study were to evaluate in vivo the effects of UC fecal supernatants and Cat-G on epithelial barrier function and inflammation, and the connection between these two parameters. METHODS: Male balb/c mice were used in this study. We evaluated the effect of a 2-hour intracolonic infusion of 1) fecal supernatants from UC patients pretreated or not with specific Cat-G inhibitor (SCGI); 2) PAR(4) -activating peptide (PAR(4) -AP); and 3) Cat-G on colonic myeloperoxidase (MPO) activity and paracellular permeability (CPP). The involvement of PAR(4) was assessed by pretreating animals with pepducin P4pal-10, which blocks PAR(4) signaling. We investigated the role of myosin light chain (MLC) kinase by using its inhibitor, ML-7, and we determined phosphorylated MLC (pMLC) levels in mice colonic mucosa. RESULTS: UC fecal supernatants, Cat-G, and PAR(4) agonist increased both CPP and MPO activity in comparison with healthy subjects fecal supernatants. ML-7 inhibited the CPP increase triggered by Cat-G by 92.3%, and the enhanced MPO activity by 43.8%. Intracolonic infusion of UC fecal supernatant determined an increased phosphorylation level of MLC. CONCLUSIONS: These observations support that luminal factors such as Cat-G play an important proinflammatory role in the pathogenesis of colitis, mainly depending on CPP increase by MLC phosphorylation.

Protease-activated receptor 1 (PAR1) signalling desensitization is counteracted via PAR4 signalling in human platelets.

PARs (protease-activated receptors) 1 and 4 belong to the family of G-protein-coupled receptors which induce both G(α12/13) and G(αq) signalling. By applying the specific PAR1- and PAR4-activating hexapeptides, SFLLRN and AYPGKF respectively, we found that aggregation of isolated human platelets mediated via PAR1, but not via PAR4, is abolished upon homologous receptor activation in a concentration- and time-dependent fashion. This effect was not due to receptor internalization, but to a decrease in Ca²âº mobilization, PKC (protein kinase C) signalling and α-granule secretion, as well as to a complete lack of dense granule secretion. Interestingly, subthreshold PAR4 activation rapidly abrogated PAR1 signalling desensitization by differentially reconstituting these affected signalling events and functional responses, which was sufficient to re-establish aggregation. The lack of ADP release and P2Y12 receptor-induced G(αi) signalling accounted for the loss of the aggregation response, as mimicking G(αi/z) signalling with 2-MeS-ADP (2-methylthioadenosine-5'-O-diphosphate) or epinephrine (adrenaline) could substitute for intermediate PAR4 activation. Finally, we found that the re-sensitization of PAR1 signalling-induced aggregation via PAR4 relied on PKC-mediated release of both ADP from dense granules and fibrinogen from α-granules. The present study elucidates further differences in human platelet PAR signalling regulation and provides evidence for a cross-talk in which PAR4 signalling counteracts mechanisms involved in PAR1 signalling down-regulation.

The pronociceptive effect of proteinase-activated receptor-4 stimulation in rat knee joints is dependent on mast cell activation.

Proteinase-activated receptor-4 (PAR(4)) is a G-protein-coupled receptor activated by serine proteinases released during tissue repair and inflammation. We have previously shown that PAR(4) activation sensitises articular primary afferents leading to joint pain. This study examined whether mast cells contribute to this PAR(4)-induced sensitisation and consequent heightened pain behaviour. The expression of PAR(4) on synovial mast cells was confirmed with immunofluorescent staining of rat knee joint sections. Electrophysiological recordings were made from joint primary afferents in male Wistar rats during both nonnoxious and noxious rotations of the knee. Afferent firing rate was recorded for 15 minutes after close intra-arterial injection of 10(-9) to 10(-5)mol of the PAR(4) activating peptide, AYPGKF-NH(2), or the inactive peptide, YAPGKF-NH(2) (100-µl bolus). Rats were either naive or pretreated with the mast cell stabilise, cromolyn (20mg/kg). Mechanical withdrawal thresholds were determined using a dynamic planter aesthesiometer and weight bearing determined using an incapacitance tester. These behavioural measurements were taken before and after intra-articular AYPGKF-NH(2), or the inactive peptide, YAPGKF-NH(2) (100µg). Local administration of AYPGKF-NH(2) caused a significant increase in joint primary afferent firing rate and pain behaviour compared with the control peptide YAPGKF-NH(2). These effects were blocked by pretreatment with cromolyn. These data reveal that PAR(4) is expressed on synovial mast cells and the activation of PAR(4) has a pronociceptive effect that is dependent on mast cell activation. Proteinase-activated receptor-4 is expressed on synovial mast cells, and the activation of Proteinase-activated receptor-4 has a pronociceptive effect that is dependent on mast cell activation.